Field of Invention
This invention generally relates to a composition and its application for producing ribonucleic acids (RNAs, i.e. messenger RNAs and microRNAs) and/or proteins/peptides (i.e. antibodies and enzymes) using eukaryotic RNA promoter-driven transcription in prokaryotes. Particularly, the present invention teaches a composition and its use for generating RNAs and/or proteins/peptides using eukaryotic type II RNA polymerase (pol-2) promoter-driven transcription in bacterial cells. Alternatively, the present invention is also an inducible gene expression composition using chemical agents rather than antibiotics to stimulate eukaryotic RNA promoter-driven transcription in prokaryotes. The novelty of the present invention is to induce a quick adaptation of prokaryotic cells to use eukaryotic pol-2 promoters for directly expressing desired RNAs and/or proteins/peptides without the need of changing to error-prone prokaryotic promoters or growing laborious and costly hybridomas or mammalian cells and to improve the reading fidelity of the transcription in the prokaryotes. The ribonucleic acids and proteins/peptides so obtained can be used to develop drugs, cure diseases, treat tumors/cancers, produce pluripotent stem (iPS) cells, enhance wound healing, and provide food supply.
Description of Related Art
As learning from current textbooks, any one of ordinary skill in the art has known very well that prokaryotic and eukaryotic transcription machineries contain differences and are not compatible to each other. For example, based on current understandings, eukaryotic RNA polymerases do not bind directly to promoter sequences and require additional accessory proteins to initiate transcription, whereas prokaryotic RNA polymerases form a holoenzyme that binds directly to promoter sequences to initiate transcription. It is also a common knowledge for an ordinary skill in the art to know that eukaryotic messenger RNA (mRNA) is synthesized in the nucleus by type II RNA polymerases (pol-2) and then processed and exported to the cytoplasm for protein synthesis, whereas prokaryotic RNA transcription and protein translation take place simultaneously off the same piece of DNA in the same place. Prokaryotes such as bacteria and archaea do not have a nucleus-like structure. These differences make a prokaryotic cell difficult or even impossible to produce eukaryotic RNAs and peptides/proteins using eukaryotic RNA promoters.
Prior art attempts at producing mammalian peptides and/or proteins in bacterial cells, such as U.S. Pat. No. 7,959,926 to Buechler and U.S. Pat. No. 7,968,311 to Mehta, used bacterial or bacteriophage promoters. For expression, the complementary DNA (cDNA) of a desired gene was cloned into a plasmid vector behind a bacterial or bacteriophage promoter. The cDNA of the desired gene must not contain any non-coding intron because bacteria do not have RNA splicing machineries to process the intron. Then, the vector so obtained was introduced into a competent strain of bacteria, such as Escherichia coli (E. coli), for expressing the desired gene transcripts (mRNAs) and further translating the mRNAs into proteins. Nevertheless, these bacterial and bacteriophage promoters, such as Tac, Lac, T3, T7, and SP6 RNA promoters, are not pol-2 promoters and their transcription is an error-prone process that tends to cause mutations. On the other hand, Mehta also taught that glycerol might be used to increase the efficiency of bacterial transformation; however, no description was related to enhancing promoter-driven RNA transcription, in particular pol-2 promoter-driven transcription. Due to lack of possible compatibility between eukaryotic and prokaryotic transcription systems, these prior arts were still limited by the use of prokaryotic RNA promoters in prokaryotes.
Traditional inducible gene expression methods, such as the old teaching from Gossen M. and Bujard H. (1992) and U.S. Pat. No. 5,464,758 to Gossen, required the use of antibiotics (e.g. tetracycline or doxycycline) to stimulate the activation and expression of a tetracycline-responsive-element (TRE)-controlled cytomegaloviral (CMV) or pol-3 (U6) promoter, namely Tet-On promoter. However, these Tet-On promoters are not eukaryotic pol-2 promoters and have never been tested in prokaryotes. Hence, if we can induce the adaptation of prokaryotic transcription machineries to express from eukaryotic pol-2 promoters, a novel inducible gene expression system will be made simply based on the differences between prokaryotic and eukaryotic transcription mechanisms rather than the previously toxic induction methods of using antibiotics, which may inhibit the growth of prokaryotes.